PURPOSES: Whether low-calorie sweeteners (LCS), such as sucralose and acesulfame K, can alter glucose metabolism is uncertain, particularly given the inconsistent observations relating to insulin resistance in recent human trials. We hypothesized that these discrepancies are accounted for by the surrogate tools used to evaluate insulin resistance and that PET 18FDG, given its capacity to quantify insulin sensitivity in individual organs, would be more sensitive in identifying changes in glucose metabolism. Accordingly, we performed a comprehensive evaluation of the effects of LCS on whole-body and organ-specific glucose uptake and insulin sensitivity in a large animal model of morbid obesity. METHODS: Twenty mini-pigs with morbid obesity were fed an obesogenic diet enriched with LCS (sucralose 1 mg/kg/day and acesulfame K 0.5 mg/kg/day, LCS diet group), or without LCS (control group), for 3 months. Glucose uptake and insulin sensitivity were determined for the duodenum, liver, skeletal muscle, adipose tissue and brain using dynamic PET 18FDG scanning together with direct measurement of arterial input function. Body composition was also measured using CT imaging and energy metabolism quantified with indirect calorimetry. RESULTS: The LCS diet increased subcutaneous abdominal fat by ≈ 20% without causing weight gain, and reduced insulin clearance by ≈ 40%, while whole-body glucose uptake and insulin sensitivity were unchanged. In contrast, glucose uptake in the duodenum, liver and brain increased by 57, 66 and 29% relative to the control diet group (P < 0.05 for all), while insulin sensitivity increased by 53, 55 and 28% (P < 0.05 for all), respectively. In the brain, glucose uptake increased significantly only in the frontal cortex, associated with improved metabolic connectivity towards the hippocampus and the amygdala. CONCLUSIONS: In miniature pigs, the combination of sucralose and acesulfame K is biologically active. While not affecting whole-body insulin resistance, it increases insulin sensitivity and glucose uptake in specific tissues, mimicking the effects of obesity in the adipose tissue and in the brain.